Dna sequence of the enzyme phospholipase a1 of ciliate tetrahymena, and the use of the same

ABSTRACT

A nucleic acid coding for the phospholipase A 1  from ciliates. In particular, the phospholipase A 1  has the amino acid sequence SEQ ID No. 7.

[0001] The invention relates to a nucleic acid coding for thephospholipase A1 and the use thereof according to the preamble of claims1 to 5.

[0002] Yeasts, bacteria and mammal cells are of great importance to thebiotechnological preparation and production of recombinant activesubstances by the heterologous expression of foreign proteins. Bacterialexpression systems based on E. coli or B. subtilis are used for theproduction of recombinant peptides or proteins, such as insulin,interleukin-2, tissue plasminogen activator, proteases and lipases. InGram-negative bacteria, the expression systems are mostly based on theuse of genetic elements, such as the lac operon or the tryptophanoperon. The proteins foreign to the host are produced either into“inclusion bodies” within the cell, or when expression systems based onβ-lactamase genes are used, into the periplasmic space. The productionof recombinant proteins into the surrounding fermentation medium has notbeen established. In Gram-Positive bacteria, to date, almost exclusivelycell-inherent proteins are introduced in expression systems andexpressed.

[0003] Yeasts, such as S. cerevisiae, Hansenula polymorpha,Kluyveromyces lactis or Pichia pastoris, are also employed for theheterologous expression of recombinant proteins, such as human factorXIIIa, bovine pro-chymosin, phytase or surface antigens. Here, theexpression systems are based on shuttle vectors (vectors having bothyeast and bacterial portions) which are based (depending on the yeastspecies) on the genetic elements of galacto-kinase-epimerase, methanoloxidase, acid phosphatase or alcohol-dehydrogenase. As a rule, therecombinant protein is produced into the cytoplasm of the cell. Whenyeast-inherent signal sequences, such as the alpha factor, are used, theexpressed proteins may also be secreted into the fermentation medium.The glycosylation of secreted proteins is effected according to the“high mannose” type, and frequently there are hyperglycosylations on theprotein which may result in the formation of antibodies in the patient.

[0004] Mammal cells, such as various cell types from rodents (CHO cells,C127 cells) or simians (vero, CV-1 or COS cells) are also employed forthe heterologous expression of recombinant proteins. Here, theexpression systems are based on recombinant viruses (BPV vector) or onshuttle vectors. To regulate the expression, viral SV40enhancer/promoter systems or cellular enhancer elements are employed.The recombinant proteins, such as erythropoietin, are secreted into thefermentation medium because the foreign genes usually bring their ownsignal sequences, which are understood by the expression system and usedfor targeting.

[0005] Further, for the biotechnological production of glycosylatedextracellular enzymes, protozoans of the genus Tetrahymena are employed.Tetrahymena will grow on inexpensive fermentation media using standardfermentation methods. For the transformation of such Tetrahymena cells,vectors are available which are based on the rDNA elements ofTetrahymena. For the heterologous expression of bacterial proteins inTetrahymena, DNA constructs made from genes from Tetrahymena areemployed. When suitable genetic elements for the regulation of thetranscription, targeting and glycosylation of foreign proteins areavailable, Tetrahymena is an ideal expression system for the inexpensiveproduction of therapeutic recombinant proteins.

[0006] The Gram-negative bacterial expression systems used to dateusually lead to the formation of “inclusion bodies” in the cell,accompanied by a denaturing of the proteins. To recover the recombinantprotein, the cells must be lysed, and the denatured inactive proteinmust be folded back to function. This causes additional cost-intensiveprocess steps and reduces the yield of the desired protein.Glycosylation, which is important to eukaryotic proteins, is completelyomitted. When Gram-positive bacterial expression systems are used,degradation of the target protein due to high proteolytic activities inthe fermentation broth is an additional problem.

[0007] When yeasts are used for heterologous expression, the desiredtarget protein is also produced only into the cell, from where it mustbe removed by cell lysis. As in bacterial expression systems, thiscauses additional time- and cost-intensive process steps. Whenyeast-inherent signal peptides are used, the foreign proteins are notcorrectly spliced and glycosylated for secretion.

[0008] In contrast, when mammal cell systems are employed for theproduction of recombinant proteins, the desired proteins are found inthe fermentation medium in an extracellular state, correctly spliced andglycosylated. However, what is disadvantageous here is, on the one hand,the low expression rate due to the defective processing and inefficienttranslation of genes which have been introduced into the genome of theproduction cell line via viral vectors. On the other hand, theserum-containing fermentation media for mammal cells are extremelycost-intensive. In addition, the fermentation technology for theshear-sensitive cell lines is complicated and similarly expensive due toconstructions for bubble-free aeration. Further problems arise from thehigh infection risk for the cell lines from mycoplasmas and viruses. Allin all, the use of mammal cells for the biotechnological preparation ofrecombinant proteins results in very high costs, safety demands and lowyields.

[0009] To the use of ciliates, such as Tetrahymena, the above mentioneddrawbacks in the production of recombinant proteins do not apply. Thus,for example, some acid hydrolases which are involved in the digestion offood particles are exported from the cell in high quantities and withcomplex glycosylation.

[0010] In J. Euk. Microbiol. 43 (4), 1996, pages 295 to 303, Alam et al.describe the cloning of a gene which codes for the acid α-glucosidase ofTetrahymena pyriformis. However, only a small portion of the protein isexported from the cell. Further, the International Patent ApplicationPCT/EP 00/01853 describes the gene of a β-hexosaminidase fromTetrahymena thermophila which is known, however, to be exported from thecell to only about 80%.

[0011] However, to date, it has not been possible to cause glycosylatedeukaryotic proteins to be expressed in Tetrahymena and also beexclusively secreted into the fermentation medium. This is because theDNA sequences of extracellular proteins inherent to Tetrahymena whichare necessary for the construction of expression vectors and whichexclusively export the foreign protein into the surrounding fermentationmedium have as yet been unknown. The DNA sequences of a protein whichcodes for the β-hexosaminidase of Tetrahymena thermophila are known.Such a sequence has been filed for a patent application under theofficial file numbers DE 199 58 979.8, DE 199 09 189.7 and under PCT/EP00/01853. However, there is a disadvantage of these sequences in thatthe pre/pro-peptides containing them will target a protein foreign tothe host into the surrounding fermentation medium to only about 80%.This is due to the fact that the enzyme β-hexosaminidase is present toabout 20% within the membrane under natural conditions, and only about80% of the naturally produced enzyme is exported from the cell. For thisreason, pre/pro peptides of β-hexosaminidase, when positioned in frontof a protein foreign to the host by genetic engineering methods, willtarget, only about 20% of this protein foreign to the host into thecytoplasma membrane on the surface of Tetrahymena thermophila. This isassociated with a considerable process-technological disadvantage forthe production of recombinant active substances. On the one hand, theyield is decreased because part of the expressed protein remains in thecells bound to the membrane, and thus it is not possible to purify theentire expressed protein from the fermenter broth. On the other hand,the protein foreign to the host in the cell membrane can exert toxiceffects on the host cells and thus slow down the cell growth.

[0012] Further, no constitutive promoters of Tetrahymena which cause aconsistent or continuous transcription of heterologous proteins havebeen known to date. To date, only promoters of histone and tubulin geneshave been known (Bannon et al., 1984, Gaertig et al., 1993). However, acritical disadvantage of these promoters is that their activation isdependent on the cell cycle. Genes of heterologous proteins which arelinked to such cell-cycle-dependent promoters are caused to be expressedonly in growing or dividing cells. This has considerableprocess-technological disadvantages since the desired protein is thusproduced only in the logarithmic growth phase. In the stationary growthphase in which the highest cell density and thus the highest performanceof the expression organism (Tetrahymena) is reached in the productionprocess, there is hardly any cell growth left and thus only a lowexpression of the heterologous protein takes place.

[0013] It is an object of the invention to provide a system whichenables the production of heterologous proteins in an expression system,after transformation into Tetrahymena, from the cells into thefermentation medium.

[0014] This object is achieved by a system in which a nucleic acidhaving the sequence SEQ ID No. 1 coding for a phospholipase A1 (SEQ IDNo. 7) is employed. Advantageously, the expression product of this DNAis exported from the cell in large amounts under culturing conditions.The expressed protein is exported into the surrounding culture medium toa high extent and is not contained in the membrane. The nucleic acidsequence according to the invention contains a promoter which causes aconstitutive, i.e., cell-cycle-independent, transcription of thedownstream genes of heterologous proteins. Such constitutivetranscription has the advantage that the proteins are continuouslyexpressed by heterologous expression in the host organism without beingaffected by the cell cycle. Thus, the transcription of the foreign genecan be effected and the heterologous protein expressed also during thestationary growth phase with a low cell growth.

[0015] The DNA sequence of phospholipase A₁ according to the inventionpreferably includes an upstream region of PLA₁ (SEQ ID No. 2) whichbears the promoter elements for the initiation of transcription, asignal peptide and a pro-peptide, further genetic elements for thetargeting of proteins and, in particular, a down-stream region of PLA₁(SEQ ID No. 3) which contains genetic elements for the termination oftranscription. The use of these nucleic acids in a vector enables theexpression of heterologously expressed proteins independently of thecell cycle and to transport them selectively out of the cell and intothe surrounding culture medium without expressed proteins becomingincorporated in the cytoplasma membrane, whereby such proteins can beisolated from the fermentation broth without cell lysis.

[0016]FIG. 1 shows a nucleic acid coding for the upstream region (SEQ IDNo. 2), the coding region (SEQ ID No. 1) and the downstream region (SEQID No. 3) of phospholipase A₁ from ciliates.

[0017]FIG. 2 shows a corresponding expression product of the nucleicacid according to SEQ ID No. 1. The invention also relates to theprotein according to SEQ ID. No. 7.

[0018] In particular, the invention also relates to the signal sequence(SEQ ID No. 6) of the protein according to the invention. Preferably,these are the amino acids 1 to 110 of the protein according to theinvention (SEQ ID No. 5). The invention also relates to a nucleic acidcoding for the N-terminal fragment (SEQ ID No. 3). This is preferably afragment of the nucleic acids according to the invention (SEQ ID No. 4),especially having the nucleic acid sequence 1 to 155 according to FIG.1.

[0019] The nucleic acid sequence of the non-translated region (upstreamregion) (SEQ ID No. 2) upstream from the coding sequence region of thePLA₁ from Tetrahymena is positioned between position −275 and position−1 (represented in lowercase letters). The established non-translatedregion comprises 275 bases. As elements of a promoter, a TATA box isfound on positions −49 to −55 (printed in boldface), and a putative CAATbox is found between base −133 and base −136 (printed in boldface). Thecoding sequence range of the cDNA is represented in capital letters. Thenumbering of the sequence begins with the start codon ATG. Regions knownfrom protein sequencing are boxed, and the stop codon is underlined. Themature protein is coded from base 331. The sequence listing from base 1to base 330 represents the pre/pro sequence (SEQ ID No. 8) of PLA₁. Thesequence listing from base 331 to base 963 is the sequence of the maturePLA₁ (SEQ ID No. 9). In position 961, there is the translation stop TGA,and in position 1039, there is the polyadenylation signal AAT AAA. Thenucleic acid sequence from position 964 to position 1134, which is belowthe coding sequence of the PLA₁ of Tetrahymena, represents thedownstream region of PLA₁ (SEQ ID No. 3) which is not translated (alsorepresented in lowercase letters). In position 964 to position 1101,there is the region known from the sequencing of the cDNA, which wasalso confirmed by inverse PCR. After transcription, the poly-A tail isattached to the last codon of the mRNA (ttt, positions 1098-1101).

[0020] A further aspect of the invention is the use of a nucleic acidsequence of acid hydrolases according to the invention or parts thereoffor the homologous or heterologous expression of recombinant proteinsand peptides, and for homologous or heterologous recombination(“knock-out, “gene replacement”).

[0021] The invention also relates to a method for the homologous orheterologous expression of proteins and peptides and for the homologousor heterologous recombination (“knock-out, “gene replacement”) in whichciliates are transfected with a nucleic acid according to the invention.

[0022] The nucleic acids or parts thereof may be combined, inparticular, with the enhancers, promoters, operators, origins,terminators, antibiotic resistances usual for the homologous orheterologous expression of proteins, or with other nucleic acids or DNAfragments or all kinds of sequences from viroids, viruses, bacteria,archezoans, protozoans, fungi, plants, animals or humans.

[0023] In particular, the nucleic acid according to the invention iscontained in a vector, a plasmid, a cosmid, a chromosome orminichromosome, a transposon, an IS element, an rDNA, or all kinds ofcircular or linear DNA or RNA.

[0024] The invention also relates to a method in which the nucleic acidor parts thereof according to the invention which code for phospholipaseA₁ are combined with the usual, in homologous or heterologousexpression, enhancers, such as the NF-1 region (a cytomegalovirusenhancer), promoters, such as the lac, trc, tic or tac promoters, thepromoters of classes II and III of the T7 RNAP system, bacteriophage T7and SP6 promoters, aprE, amylase or spac promoters for Bacillusexpression systems, AOX1, AUG1 and 2 or GAPp promoters (Pichia) foryeast expression systems, RSV promoter (SV40 virus), CMV promoter(Cytomegalovirus), AFP promoter (adenoviruses) or metallothioninepromoters for mammal expression systems, Sindbis virus promoters orSemlike forest virus promoters for insect cells, promoters for insectcell expression systems, such as hsp70, DS47, actin 5C or copia,plant-specific promoters, such as 35S promoter (cauliflower mosaicvirus), amylase promoter or class I patatin promoter, operators, such asthe tet operator, signal peptides, such as a-MF prepro signal sequences(Saccharomyces), origins, terminators, antibiotic and drug resistances,such as ampicillin, kanamycin, streptomycin, chloramphenicol,penicillin, amphotericin, cycloheximide, 6-methylpurine, paromomycin,hygromycin, α-amanatin, auxotrophy markers, such as the gene ofdihydrofolate reductase, or other nucleic acids or DNA fragments, or allkinds of sequences from viroids, viruses, bacteria, archezoans,protozoans, fungi, plants, animals or humans.

[0025] In particular, the nucleic acid or parts thereof according to theinvention are inserted into a vector, a plasmid, a cosmid, a chromosomeor minichromosome, a transposon, an IS element, an rDNA, or all kinds ofcircular or linear DNA or RNA.

[0026] The skilled person will understand that nucleic acids having atleast 40% homology with the nucleic acid according to SEQ ID No. 1 canalso be employed according to the invention. The protein according toSEQ ID No. 2 can also be modified without losing its function. Thus, forexample, so-called conservative exchanges of amino acids may beperformed. Thus, for example, hydrophobic amino acids can beinterchanged.

[0027] For the purification and isolation of phospholipase A₁ fromTetrahymena and for determining its sequence, the following methods canbe used.

[0028] Recovery of PLA₁

[0029] PLA1 was obtained from cell-free culture supernatants ofTetrahymena thermophila. Thus, the cells were fermented in a 2 Ifermenter (Biostat M D, Braun Diessel Biotech, Melsungen, Germany) whichwas controlled over a digital controlling unit (DCU). The fermenter wasfirst operated for 24 hours in a batch operation and then continuously.Harvesting of the cell-free culture supernatant was ensured through aperfusion module having a pore size of about 0.3 μm (S6/2, Enka,Wuppertal).

[0030] The fermentation was performed under the following parameters:

[0031] the working volume was 2 liters;

[0032] the perfusion rate was 2 liters/day;

[0033] the revolutions per minute of the stirrer was limited to 800 rpm;

[0034] the temperature was constantly at 30° C.;

[0035] the pH value was kept constant at pH 7;

[0036] the inoculation titer was at 50,000 cells/ml.

[0037] For the fermentation, the strain SB 1868 VII was used. This is awild type strain of Tetrahymena thermophila.

[0038] The fermentation was performed over a period of 264 hours, andthe harvests were tested for PLA₁ activity.

[0039] Purification of PLA₁

[0040] For the purification of PLA₁, 1 liter of cell-free culturesupernatant from the fermentation was used. It was admixed with 140 g ofammonium sulfate and concentrated through an ultrafiltration unit(Pellikon XL, exclusion size 3 kDa, Millipore) to a volume of 50 ml.Subsequently, the sample was purified by hydrophobic interactionchromatography (20×1,6 Fractogel EMD Phenyl I 650, Merck, Darmstadt).The flow rate was 5 ml/min, and the eluate was collected in 5 mlfractions. The enzyme activity was measured by the deacylation of aradioactively labeled phospholipid (L-3-phosphatidylcholine,1-palmitoyl-2-[1-¹⁴C]linoleoyl). FIG. 3 shows the elution profileobtained, the sodium acetate gradient and the enzyme activities in theindividual fractions.

[0041] The three fractions having the highest enzyme activities werecombined and rebuffered into the starting buffer (Bis-Tris 20 mM, pH6.5) for anion-exchange chromatography (AEC) by means of anultrafiltration unit. Subsequently, the sample was charged onto thecolumn (Q-Sepharose-Hiload-16/10, Pharmacia, Sweden), and the PLA₁ waseluted with a linear NaCl gradient (flow rate=3 ml/min) from the columnand collected in 5 ml fractions. FIG. 4 shows the elution profileobtained, the NaCl gradient and the enzyme activities of the individualfractions.

[0042] From the fraction having the highest PLA₁ activity, 200 μl wasremoved and separated by size exclusion chromatography (SEC). For thispurpose, a Superdex HR 75 30/10 column (Pharmacia, Sweden) was used. Theflow rate in this chromatography was 0.6 ml/min, the eluate wascollected in 200 μl fractions. FIG. 5 shows the elution profile obtainedand the enzyme activities of the individual fractions.

[0043] The fractions obtained were examined for their purity usingone-dimensional gel electrophoresis. Thus, two distinct bands wereestablished at ˜26 and ˜28 kDa. Separation of these two bands by atwo-dimensional gel electrophoresis resulted in a separation of the twobands into 2 and 3 spots, respectively, having different isoelectricpoints.

[0044] For the 26 kDa proteins, these were at pH 6.3 and 5.7, and forthe 28 kDa proteins, they were at pH 6.3, 5.7 and 5.3. A finalexamination of these spots by mass fingerprint analysis showed, thatthese spots were isoforms of the same protein.

[0045] Molecular-Biological Examination of PLA₁

[0046] After the purity of the protein had been demonstrated, samples ofthe protein were blotted onto a PVDF membrane and subjected to initialsequencing from the N terminus. In addition, a further sample wastryptically digested and also subjected to initial sequencing. Using theprotein sequences obtained thereby, oligonucleotide primers wereprepared, which were then employed in reverse transcriptase PCR (3′RACE, rapid amplification of cDNA ends). Using this PCR technique, cDNAof phospholipase A₁ was successfully amplified and subsequentlysequenced. The sequence obtained had a length of 633 bases and 729bases, respectively, and the molecular weight of the mature proteinderived therefrom is about 22.4 kDa. In the sequence derived, theoligopeptides of 22 amino acids (N-terminal) and 18 amino acids (withinthe protein) established from protein sequencing were found again to100%. In addition to the sequence of the mature protein, the sequence ofthe pre/pro peptide could also be established by means of 5′ RACE (rapidamplification of cDNA ends) (FIG. 2). This is a peptide having a lengthof 110 amino acids which bears both the signal sequence and the propeptide which inactivates the enzyme and is cleaved off only at thefinal place of activity of the enzyme.

[0047] Sequence comparisons yielded no homologies with previously knownphospholipases A₁, except for a consensus sequence of 5 amino acids(G×S×G), which is found in all lipases and phospholipases and isdiscussed as a binding site for lipids or phospholipids. Further, theupstream and downstream sequences of PLA₁ were established by inversePCR (FIG. 1). Thus, genomic DNA was cut with restriction endonucleases,ligated with T4 ligase and finally amplified with inverse primers. Forthe amplification of the upstream region of PLA₁ by inverse PCR, genomicDNA cut with the restriction endonuclease SspI was used. Thus, anupstream region of 275 bases could be established, and promoter elementsidentified. In position −136, there is a CAAT box which has a similardistance from the translation start as the CCAAT boxes of the histonegenes (−141 and −151) of Tetrahymena as found by Brunk and Sadler(1990). A TATA box, which fixes the exact starting point oftranscription in eukaryotic genes, was identified on position −55. Itssequence corresponds to the consensus sequences found in eukaryotes. Forthe amplification of the downstream region, which contains theterminator for the transcription of the PLA₁ from Tetrahymena, byinverse PCR, genomic DNA cut with BamHI was used. Thus, in addition tothe downstream region known from 3′-RACE, another 222 bases could beestablished (FIG. 3).

1 9 1 963 DNA Tetrahymena thermophila 1 atgaacaaga ctctcatctt agctttagttgttgttttgg ctttaactgc caccaccttg 60 gttgctttcc acaaccactc tcacaacatcagagttgact aagaccccgc cactctcttc 120 aagcaattca agcaaactta caataagaagtatgctgatg ctactttcga aacctacaga 180 ttcggtgtct tcacccaaaa cttagaaatcgtcaagactg actctacttt cggtgtcacc 240 taattcatgg acttaactcc tgctgaattcgctcaacaat tcctcacttt acacgaaaag 300 gttaacagca ccgaagttta cagagcttaaggtgaagcta ccgaagttga ctggactgcc 360 aagggtaagg tcacccctgt taagaactaaggttcttgtg gttcctgctg ggctttctcc 420 accattggtg ccgttgaatc tgctctttggattgctggtc aaggtgaata aaacactctt 480 aaccttgctg aataagaata agttgactgtgctaagtccc ccaagtacga ctctgaaggt 540 tgcaacggtg gttggatggt tgaaggtttcaagtacatca tcgacaacaa gatctcctaa 600 actgctaact atccctacac tgctaaggatggtaagtgca aggacacctc ttccttcaag 660 aagttctcta tttctaagta cgctgaaatcccctaaggtg actgcaactc cctcaactct 720 gccttagaac aaggtcctat ctccgttgctgttgatgcca ccaacttcta attctacact 780 tctggtgtct ttaaaaactg caaggccaacctcaaccacg gtgtcctctt agttgccaac 840 gttgactctt ctctcaagat caagaactcctggggtcctt cttggggtga aaagggtttc 900 atcagattag ctgccggtaa cacttgcggtgtctgcaatg ctgcctctta ccctattgtt 960 tga 963 2 275 DNA Tetrahymenathermophila 2 aatatttatc aatgctactt ataattcttt tagtatgaga tatgatatgctctttctctg 60 ctagacttaa cttatgacat ttgaactttt aataaaagaa ttttttttattaaaaagcag 120 agatttttaa tagaagaatc aatgactcat gaatttaata aagattttcaagtgttttct 180 aataccgact agctttataa attcacttat taatcaacga tataaaaattatattaacaa 240 atcaataaat aaaaaaataa ataaaaacaa aacaa 275 3 171 DNATetrahymena thermophila 3 aaaaacataa tccaaattaa aaaaaattac tcaaaactgataatataaaa aattaatttt 60 cataatttta atgtaaataa atacctttat atttgacgttttgtactcaa aataaattaa 120 agttaacaaa ccatatttat ttaattctac ttttcaatttttaaaaatat a 171 4 1408 DNA Tetrahymena thermophila 4 aatatttatcaatgctactt ataattcttt tagtatgaga tatgatatgc tctttctctg 60 ctagacttaacttatgacat ttgaactttt aataaaagaa ttttttttat taaaaagcag 120 agatttttaatagaagaatc aatgactcat gaatttaata aagattttca agtgttttct 180 aataccgactagctttataa attcacttat taatcaacga tataaaaatt atattaacaa 240 atcaataaataaaaaaataa ataaaaacaa aacaaatgaa caagactctc atcttagctt 300 tagttgttgttttggcttta actgccacca ccttggttgc tttccacaac cactctcaca 360 acatcagagttgactaagac cccgccactc tcttcaagca attcaagcaa acttacaata 420 agaagtatgctgatgctact ttcgaaacct acagattcgg tgtcttcacc caaaacttag 480 aaatcgtcaagactgactct actttcggtg tcacctaatt catggactta actcctgctg 540 aattcgctcaacaattcctc actttcacga aaaggttaac agcaccgaag tttacagagc 600 ttaaggtgaagctaccgaag ttgactggac tgccaagggt aaggtcaccc ctgttaagaa 660 ctaaggttcttgtggttcct gctgggcttt ctccaccatt ggtgccgttg aatctgctct 720 ttggattgctggtcaaggtg aataaaacac tcttaacctt gctgaataag aataagttga 780 ctgtgctaagtcccccaagt acgactctga aggttgcaac ggtggttgga tggttgaagg 840 tttcaagtacatcatcgaca acaagatctc ctaaactgct aactatccct acactgctaa 900 ggatggtaagtgcaaggaca cctcttcctt caagaagttc tctatttcta agtacgctga 960 aatcccctaaggtgactgca actccctcaa ctctgcctta gaacaaggtc ctatctccgt 1020 tgctgttgatgccaccaact tctaattcta cacttctggt gtctttaaaa actgcaaggc 1080 caacctcaaccacggtgtcc tcttagttgc caacgttgac tcttctctca agatcaagaa 1140 ctcctggggtccttcttggg gtgaaaaggg tttcatcaga ttagctgccg gtaacacttg 1200 cggtgtctgcaatgctgcct cttaccctat tgtttgaaaa aacataatcc aaattaaaaa 1260 aaattactcaaaactgataa tataaaaaat taattttcat aattttaatg taaataaata 1320 cctttatatttgacgttttg tactcaaaat aaattaaagt taacaaacca tatttattta 1380 attctacttttcaattttta aaaatata 1408 5 80 PRT Tetrahymena thermophila 5 Ala Leu GluGln Gly Pro Ile Ser Val Ala Val Asp Ala Thr Asn Phe 1 5 10 15 Gln PheTyr Thr Ser Gly Val Phe Lys Asn Cys Lys Ala Asn Leu Asn 20 25 30 His GlyVal Leu Leu Val Ala Asn Val Asp Ser Ser Leu Lys Ile Lys 35 40 45 Asn SerTrp Gly Pro Ser Trp Gly Glu Lys Gly Phe Ile Arg Leu Ala 50 55 60 Ala GlyAsn Thr Cys Gly Val Cys Asn Ala Ala Ser Tyr Pro Ile Val 65 70 75 80 6110 PRT Tetrahymena thermophila 6 Met Asn Lys Thr Leu Ile Leu Ala LeuVal Gly Val Leu Ala Leu Thr 1 5 10 15 Ala Thr Thr Leu Val Ala Phe HisAsn His Ser His Asn Ile Arg Val 20 25 30 Asp Gln Asp Pro Ala Thr Leu PheLys Gln Phe Lys Gln Thr Tyr Asn 35 40 45 Lys Lys Tyr Ala Asp Pro Thr PheGlu Thr Tyr Arg Phe Gly Val Phe 50 55 60 Thr Gln Asn Leu Glu Ile Val LysThr Asp Ser Thr Phe Gly Val Thr 65 70 75 80 Gln Phe Met Asp Leu Thr ProAla Glu Phe Ala Gln Gln Phe Leu Thr 85 90 95 Leu His Glu Lys Val Asn SerThr Glu Val Tyr Arg Ala Gln 100 105 110 7 210 PRT Tetrahymenathermophila 7 Gly Glu Ala Thr Glu Val Asp Trp Thr Ala Lys Gly Lys ValThr Pro 1 5 10 15 Val Lys Asn Gln Gly Ser Cys Gly Ser Cys Trp Ala PheSer Thr Ile 20 25 30 Gly Ala Val Glu Ser Ala Leu Leu Ile Ala Gly Gln GlyGlu Gln Asn 35 40 45 Thr Leu Asn Leu Ala Glu Gln Glu Leu Val Asp Cys AlaLys Ser Pro 50 55 60 Lys Tyr Asp Ser Glu Gly Cys Asn Gly Gly Trp Met ValGlu Gly Phe 65 70 75 80 Lys Tyr Ile Ile Asp Asn Lys Ile Ser Gln Thr AlaAsn Tyr Pro Tyr 85 90 95 Thr Ala Lys Asp Gly Lys Cys Lys Asp Thr Ser SerPhe Lys Lys Phe 100 105 110 Ser Ile Ser Lys Tyr Ala Glu Ile Pro Gln GlyAsp Cys Asn Ser Leu 115 120 125 Asn Ser Ala Leu Glu Gln Gly Pro Ile SerVal Ala Val Asp Ala Thr 130 135 140 Asn Phe Gln Phe Tyr Thr Ser Gly ValPhe Lys Asn Cys Lys Ala Asn 145 150 155 160 Leu Asn His Gly Val Leu LeuVal Ala Asn Val Asp Ser Ser Leu Lys 165 170 175 Ile Lys Asn Ser Trp GlyPro Ser Trp Gly Glu Lys Gly Phe Ile Arg 180 185 190 Leu Ala Ala Gly AsnThr Cys Gly Val Cys Asn Ala Ala Ser Tyr Pro 195 200 205 Ile Val 210 8330 DNA Tetrahymena thermophila 8 atgaacaaga ctctcatctt agctttagttgttgttttgg ctttaactgc caccaccttg 60 gttgctttcc acaaccactc tcacaacatcagagttgact aagaccccgc cactctcttc 120 aagcaattca agcaaactta caataagaagtatgctgatg ctactttcga aacctacaga 180 ttcggtgtct tcacccaaaa cttagaaatcgtcaagactg actctacttt cggtgtcacc 240 taattcatgg acttaactcc tgctgaattcgctcaacaat tcctcacttt acacgaaaag 300 gttaacagca ccgaagttta cagagcttaa330 9 633 DNA Tetrahymena thermophila 9 ggtgaagcta ccgaagttga ctggactgccaagggtaagg tcacccctgt taagaactaa 60 ggttcttgtg gttcctgctg ggctttctccaccattggtg ccgttgaatc tgctctttgg 120 attgctggtc aaggtgaata aaacactcttaaccttgctg aataagaata agttgactgt 180 gctaagtccc ccaagtacga ctctgaaggttgcaacggtg gttggatggt tgaaggtttc 240 aagtacatca tcgacaacaa gatctcctaaactgctaact atccctacac tgctaaggat 300 ggtaagtgca aggacacctc ttccttcaagaagttctcta tttctaagta cgctgaaatc 360 ccctaaggtg actgcaactc cctcaactctgccttagaac aaggtcctat ctccgttgct 420 gttgatgcca ccaacttcta attctacacttctggtgtct ttaaaaactg caaggccaac 480 ctcaaccacg gtgtcctctt agttgccaacgttgactctt ctctcaagat caagaactcc 540 tggggtcctt cttggggtga aaagggtttcatcagattag ctgccggtaa cacttgcggt 600 gtctgcaatg ctgcctctta ccctattgtttga 633

1. A nucleic acid coding for the phospholipase A₁ from ciliates.
 2. Thenucleic acid according to claim 1, comprising a coding region andnon-coding regions.
 3. The nucleic acid according to claim 2, whereinthe nucleic acid fragments of the non-coding regions have the SEQ IDNos. 2 and 3, and the nucleic acid of the coding region has the SEQ IDNos. 1, 8 and/or
 9. 4. A nucleic acid consisting of coding andnon-coding regions and having the SEQ ID No.
 4. 5. A protein having theamino acid sequence Fig. SEQ ID No.
 5. 6. An N-terminal fragment of theprotein according to claim 5, having the sequence SEQ ID No.
 6. 7. Aphospholipase A₁ protein (PLA₁) having the amino acid sequence accordingto SEQ ID No.
 7. 8. Use of a nucleic acid according to claims 1 to 3 orparts thereof for the homologous or heterologous expression ofrecombinant proteins and peptides, and for homologous or heterologousrecombination (“knock-out, “gene replacement”).
 9. A method for thehomologous or heterologous expression of proteins and peptides and forthe homologous or heterologous recombination (“knockout, “genereplacement”) in which ciliates are transfected with a nucleic acidaccording to any of claims 1 to
 3. 10. The method according to claim 9,wherein said nucleic acids or parts thereof are combined with theenhancers, promoters, operators, origins, terminators, antibioticresistances usual for the homologous or heterologous expression ofproteins, or with other nucleic acids or DNA fragments or all kinds ofsequences from viroids, viruses, bacteria, archezoans, protozoans,fungi, plants, animals or humans.
 11. The method according to claim 10and/or 11, wherein a nucleic acid is contained in a vector, a plasmid, acosmid, a chromosome or minichromosome, a transposon, an IS element, anrDNA, or any other kind of circular or linear DNA or RNA.
 12. Vectors,plasmids, cosmids, chromosomes or minichromosomes, transposons, ISelements, rDNA or other kinds of circular or linear DNA or RNAcontaining at least one of the nucleic acids according to at least oneof claims 1 to 3.